Current vaccines against Haemophilus influenzae type b reduced the incidence of H. influenzae invasive diseases. However, nontypeable H. influenzae are a common cause of pneumonia and otitis media, which is associated with hearing loss and language deficits. H. influenzae has an absolute growth requiremnet for heme and the human body is its sole niche. Previously, we characterized a family of genes encoding proteins, Hgp, which bind human hemoglobin and the hemoglobin-haptoglobin complex. Mutation of all the hgp genes in a strain does not abrogate hemoglobin binding. We have identified a putative gene product encoding this residual hemoglobin utilization activity. Expression of Hgps is repressible by heme, but not by elemental iron. The upstream region of each gene lacks the Fur consensus site suggesting that the Fur repressor does not directly regulate the Hgps. We have isolated H. influenzae fur mutants and fur-independent mutants with altered hgp expression. Thus, regulation is more complex thatn the classic ferric uptake repressor system described for a wide range of bacterial species. Sequence analysis of Hgps reveals four highly conserved regions. Preliminary data about the complex regulation of hgp expression, localization of a binding region of HgpA, and the identification of conserved regions provide an opportunity to investigate the structure/function, gene regulation, and immunobiology of the Hgps. The current project will determine ligands bound by Hgp, identify binding sites, characterize the conserved regions, and determine the relative growth advantage of multiple Hgps in a strain. In addition, the role of fur and fur-independent elements in the regulation of Hgp will be examined. Finally characterization of the immunobiology of the Hgps will determine the protective capacity of antisera to the conserved regions of the Hgps in animal models of invasive and noninvasive disease. These experiments will provide insight into how heme acquisition is related to pathogenicity. These studies will lay the foundation for long-term studies focusing on prevention of all H. influenzae related disease.